ObjectiveTo investigate the knockdown efficiency of the liver-specific knockdown recombinant adeno-associated virus （AAV） thyroid-binding globulin （TBG） （AAV-TBG-shOTUD6B） targeting the ovarian tumor domain 6B （OTUD6B） of the ubiquitin-proteasome system in the liver of C57BL/6J mice and its regulatory effect on liver glucose metabolism. MethodsAn effective siOTUD6B sequence was designed and verified. The DNA sequences of non-specific interference RNA negative control （shNC） and shOTUD6B were inserted into the AAV vector backbone containing the promoter TBG to construct and purify AAV-TBG-shNC and AAV-TBG-shOTUD6B viruses. AAV-TBG-shNC and AAV-TBG-shOTUD6B were injected into mice via the tail vein to target the liver. After 8 weeks of high-fat diet （HFD） feeding， the levels of OTUDB protein and mRNA in the mice liver， as well as blood glucose levels， glucose tolerance， insulin sensitivity， and protein expression levels of phosphorylated protein kinase B （p-AKT） and phosphorylated glycogen synthase kinase-3β （p-GSK-3β） were detected. ResultsCompared with the AAV-TBG-shNC group， the OTUD6B protein level in the liver of the AAV-TBG-shOTUD6B group decreased， the mRNA level of OTUD6B in the liver decreased （P<0.01）， the fasting blood glucose levels of the mice after 4 hours of fasting and 16 hours of fasting both decreased （P<0.05， P<0.01）， the protein level of p-GSK-3β in the liver decreased （P<0.05）， glucose tolerance was enhanced （P<0.05）， insulin sensitivity was enhanced （P<0.01）， and the protein level of p-AKT in the liver increased （P<0.05）. ConclusionThe construction of a recombinant adeno-associated virus for liver OTUD6B knockdown is successfully completed. OTUD6B plays an important role in regulating the homeostasis of liver glucose metabolism. Liver-specific knockdown of OTUD6B can improve the blood glucose levels of mice induced by HFD and enhance insulin sensitivity.